title: Increased stromal extracellular matrix synthesis and assembly by insulin activated bovine keratocytes cultured under agarose.
abstract: Previously, pharmacological levels of insulin have been shown to stimulate the synthesis of normal corneal stromal collagen and proteoglycans by bovine keratocytes in culture. Here we compared insulin to physiological levels of IGF-I and found that IGF-I also stimulated the synthesis of these extracellular matrix components, but less than that of insulin. Keratocytes in monolayer culture secreted most of the collagen synthesized into the media in the form of procollagen, a precursor of collagen. We found that an overlay of 3% agarose on the keratocytes in culture enhanced the conversion of procollagen to collagen and increased the deposition of collagen and proteoglycans into the cell layer. The extracellular matrix associated with the keratocytes cultured under agarose exhibited a corneal stromal-like architecture. These results suggest that enhancing the conversion of procollagen to collagen is a key step in the formation of extracellular matrix by keratocytes in vitro. Agarose overlay of insulin activated keratocytes in culture is a useful model for studying corneal stromal extracellular matrix assembly in vitro.
authors.affiliation: Department of Molecular Medicine, College of Medicine, University of South Florida, Tampa, FL 33612-4799, USA. jhassell@health.usf.edu
authors.forename: John R
authors.initials: JR
authors.lastname: Hassell
authors.forename: Bradley P
authors.initials: BP
authors.lastname: Kane
authors.forename: La Tia
authors.initials: LT
authors.lastname: Etheredge
authors.forename: Nikola
authors.initials: N
authors.lastname: Valkov
authors.forename: David E
authors.initials: DE
authors.lastname: Birk
mesh_terms: D000818:Animals; D002417:Cattle; D018929:Cell Culture Techniques; D049109:Cell Proliferation / Q000187:drug effects; D003094:Collagen / Q000096:biosynthesis; D003319:Corneal Stroma / Q000187:drug effects* / Q000378:metabolism / Q000648:ultrastructure; D003470:Culture Media; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D005109:Extracellular Matrix / Q000378:metabolism* / Q000648:ultrastructure; D007328:Insulin / Q000494:pharmacology*; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology; D008854:Microscopy, Electron; D011509:Proteoglycans / Q000096:biosynthesis; D012685:Sepharose; D014617:Vacuoles / Q000648:ultrastructure
chemical_list: D003470:Culture Media; D007328:Insulin; D011509:Proteoglycans; D007334:Insulin-Like Growth Factor I; D003094:Collagen; D012685:Sepharose